Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical‐microbead‐based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high‐aspect‐ratio hydrogel microstrands are introduced to overcome these limitations. Pre‐crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co‐printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.

Tissue engineering strongly relies on the use of hydrogels as highly hydrated 3D matrices to support the maturation of laden cells. However, because of the lack of microarchitecture and sufficient porosity, common hydrogel systems do not provide physical cell-instructive guidance cues and efficient transport of nutrients and oxygen to the inner part of the construct. A controlled, organized cellular alignment and resulting alignment of secreted ECM are hallmarks of muscle, tendons, and nerves and play an important role in determining their functional properties. Although several strategies to induce cellular alignment have been investigated in 2D systems, the generation of cell-instructive 3D hydrogels remains a challenge. Here, we report on the development of a simple and scalable method to efficiently generate highly macroporous constructs featuring aligned guidance cues. A precross-linked bulk hydrogel is pressed through a grid with variable opening sizes, thus deconstructing it into an array of aligned, high aspect ratio microgels (microstrands) with tunable diameter that are eventually stabilized by a second photoclick cross-linking step. This method has been investigated and optimized both in silico and in vitro, thereby leading to conditions with excellent viability and organized cellular alignment. Finally, as proof of concept, the method has been shown to direct aligned muscle tissue maturation. These findings demonstrate the 3D physical guidance potential of our system, which can be used for a variety of anisotropic tissues and applications.

31Hydrogels are an excellent biomimetic of the extracellular matrix and have found great 32 use in tissue engineering. Nanoporous monolithic hydrogels have limited mass transport, 33 restricting diffusion of key biomolecules. Structured microbead-hydrogels overcome some 34 of these limitations, but suffer from lack of controlled anisotropy. Here we introduce a 35 novel method for producing architected hydrogels based on entanglement of microstrands. 36 The microstrands are mouldable and form a porous structure which is stable in water. 37 Entangled microstrands are useable as bioinks for 3D bioprinting, where they align during 38 the extrusion process. Cells co-printed with the microstrands show excellent viability and 39 augmented matrix deposition resulting in a modulus increase from 2.7 kPa to 780.2 kPa 40 after 6 weeks of culture. Entangled microstands are a new class of bioinks with 41 unprecedented advantages in terms of scalability, material versatility, mass transport, 42 showing foremost outstanding properties as a bioink for 3D printed tissue grafts. 43 44 45 46 47 48 49 50 129 130 131 Results 132 133 Entangled Microstrand Materials are Mouldable, Stable in Water and Macroporous 134 135Here we report on a robust and versatile method for preparing 'entangled' microstrands. 136 Bulk hyaluronan-methacrylate (HA-MA) hydrogels were mechanically pressed through a 137

In recent years, the development of novel photocrosslinking strategies and photoactivatable materials has stimulated widespread use of light‐mediated biofabrication techniques. However, despite great progress toward more efficient and biocompatible photochemical strategies, current photoresins still rely on photoinitiators (PIs) producing radical‐initiating species to trigger the so‐called free‐radical crosslinking/polymerization. In the context of bioprinting, where cells are encapsulated in the bioink, the presence of radicals raises concerns of potential cytotoxicity. In this work, a universal, radical‐free (RF) photocrosslinking strategy to be used for light‐based technologies is presented. Leveraging RF uncaging mechanisms and Michael addition, cell‐laden constructs are photocrosslinked by means of one‐ and two‐photon excitation with high biocompatibility. A hydrophilic coumarin‐based group is used to cage a universal RF photocrosslinker based on 4‐arm‐PEG‐thiol (PEG4SH). Upon light exposure, thiols are uncaged and react with an alkene counterpart to form a hydrogel. RF photocrosslinker is shown to be highly stable, enabling potential for off‐the‐shelf products. While PI‐based systems cause a strong upregulation of reactive oxygen species (ROS)‐associated genes, ROS are not detected in RF photoresins. Finally, optimized RF photoresin is successfully exploited for high resolution two‐photon stereolithography (2P‐SL) using remarkably low polymer concentration (<1.5%), paving the way for a shift toward radical‐free light‐based bioprinting.

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